A dynamic cycle of O-linked N-acetylglucosamine (O-GlcNAc) addition and removal acts on nuclear pore proteins, transcription factors, and kinases to modulate cellular signaling cascades. This nutrient sensing hexosamine signaling pathway is conserved from nematodes to man. A single nucleotide polymorphism in the human O-GlcNAcase gene is linked to type 2 diabetes, suggesting that perturbation of this pathway results in disease.[unreadable] [unreadable] In collaboration the Hanover lab (NIDDK), we showed that the C. elegans genome encodes the two evolutionarily conserved enzymes that mediate O-GlcNAc cycling, with the genes called ogt-1 and oga-1. We previously characterized a knockout allele of ogt-1 gene. Now, we demonstrate that oga-1 encodes an active O-GlcNAcase when expressed in E. coli and we describe a knockout allele, oga-1 (ok1207), that is viable and fertile yet accumulates O-GlcNAc on nuclear pores and other cellular proteins. Interfering with O-GlcNAc recycling with either oga-1(ok1207) or the O-GlcNAc transferase null ogt-1(ok430) globally altered Ser- and Thr-phosphorylation and specifically increased phospho-GSK-3 levels. Like ogt-1(ok430), the oga-1(ok1207) strain exhibited elevated glycogen and trehalose stores and decreased lipid storage suggesting that the insulin-like signaling pathway controlling storage, longevity and dauer formation was perturbed by inhibiting O-GlcNAc cycling. Furthermore, the oga-1(ok1207) knockout augmented an alternate developmental life cycle (dauer formation) as assayed by a temperature sensitive allele of the insulin-like receptor (daf-2). Under the same conditons, the ogt-1(ok430) mutant diminished dauer formation. Our findings suggest that the highly conserved enzymes of O-GlcNAc recycling fine-tune insulin-like signaling in C. elegans in response to nutrient flux. The knockout of O-GlcNAcase in C. elegans mimics many of the metabolic and signaling changes associated with human insulin resistance and may provide a genetically amenable model of non-insulin dependent diabetes.